Specific suppression of the host's immune response to donor HLA antigens remains the ultimate goal for clinical transplantation. In spite of considerable effort, however, allospecific human suppressor T cells (Ts) have been difficult to generate. The studies herein (first series of experiments) show that allospecific and xenospecific Ts can be raised by multiple priming of human T cells in mixed lymphocyte cultures (MLC). Ts derive from the CD8+CD28− subset and recognize specifically the MHC class I antigens expressed by Antigen-Presenting Cells (APC) used for in vitro immunization. Allospecific Ts prevent the upregulation of B7 molecules on target APCs, interfering with the CD28-B7 interaction required for T helper (Th) activation. These findings provide a basis for the development of specific immunosuppressive therapy.
The induction of donor-specific tolerance remains the ultimate goal for clinical transplantation. Immunosuppressive treatments that have been developed so far act non-specifically, placing the recipient at increased risk for infections and malignancies.
Transplant tolerance has been induced in adult animals by inactivation or depletion of mature T lymphocytes prior to transplantation using cyclosporine (CsA) (1), total lymphoid irradiation (2,3), anti-lymphocyte serum (4), antibodies against CD4+ and CD8+ T cells (5), or donor-specific transfusions (6,7). Studies of peripheral graft tolerance have suggested the existence of an active mechanism of suppression which is donor-specific and can be transferred adoptively to secondary hosts (1, 7–10). However, there is still controversy concerning the phenotypic characteristics of these regulatory T cells and their MHC restriction, as both CD8− and CD4+ T cells were reported to display suppressive activity (11). This controversy lead to the speculation that no distinctive Ts lineage actually exists. It has been suggested that suppression may result from antagonistic effects of (Th)2-type lymphokines (such as IL-4 and IL-10) on the response of Th1 cells (2,12), or from recognition by Ts of either idiotypic determinants of the TCR of alloreactive T cells or of MHC antigens expressed on stimulating cells (10,13). The generation of Ts lines has proven, however, to be a difficult task rendering the characterization of these cells hard to achieve.
The aim of the present study (first series of experiments) was to develop and characterize suppressor T cell lines which inhibit specifically the alloimmune response. This study established for the first time the existence of a population of CD8+CD28− Ts which are allorestricted by HLA-class I antigens expressed by the cells used for priming. The mechanism of suppression is based on the capacity of Ts to prevent the upregulation of B7 molecules (CD80 and CD86) induced by Th on the stimulating APC. Allorestricted Ts can be easily and reproducibly expanded in cultures facilitating the in vitro study of immunoregulatory networks and the development of new strategies for specific immunosuppression.
Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or T helper cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. In the second series of experiments herein it is shown that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCS. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28− phenotype and react to MHC class I antigens expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vβ families, indicating that xenostimulation of CD8+CD28− T cells results in antigen-driven selection of a limited Vβ repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of T helper (Th) cells, inhibiting their ability to react against the immunizing MHC-class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.
The induction of regulatory T cells may offer an effective means for specific immunosuppression of autoimmune disease and allograft rejection. The existence of suppressor T cells has been previously documented, yet their mechanism of action remains poorly characterized. The third series of studies herein demonstrate that T suppressor (Ts) cell lines can be generated by in vitro immunization of human PBMCs, with synthetic peptides or soluble proteins coupled to beads. Such Ts cells express the CD8+CD28− phenotype and show the following characteristics: a) antigen specificity and restriction by self MHC Class I molecules, b) limited TCR V beta gene usage, c) ability to inhibit antigen-specific, MHC Class II restricted, Th proliferative responses, and d) capacity to downregulate and/or inhibit the upregulation by Th of CD40, CD80, and CD86 molecules on APCs. The inhibitory activity of Ts on Th proliferation requires the tripartite interaction between Th, Ts, and APCs and results from inefficient costimulation of Th.
Understanding the mechanism which underlies the induction of immunologic tolerance is crucial to the development of strategies for treatment of auto-immune diseases and allograft rejection. Although the concept that T suppressor cells (Ts) downregulate the immune response has long been accepted, the existence of a distinct population of lymphocytes that mediates suppression has not been convincingly demonstrated. In previous studies, human T cell lines (TCLs) were utilized to analyze the suppressive effects of CD8+ CD28− T cells in allogeneic, peptide specific and xeno-specific responses. In each case, CD8+ CD28− T cells inhibit proliferation of CD4+ T helper lymphocytes (Th) with cognate antigen specificity. These CD8+ CD28− T cells display the critical functional characteristics of T suppressor cells. Similar to the induction of CD8+ cytotoxic T cells (Tc) by Th, this process depends on antigen presenting cells (APC) acting as a “bridge” between MHC-class I specific CD8+ and class II specific CD4+ T cells. A possible explanation of Ts-mediated suppression is their ability to modulate the function of APCs. The fourth series of studies herein show that CD8+CD28− Ts directly inhibit the CD40 signaling pathway of APC by a contact-dependent mechanism that renders bridging APCs incapable of inducing CD4+ The activation. The effects of Ts on the functional state of APC supports the concept that the order in which Ts and Th cells interact with cognate APCs determines the functional outcome of immune responses.